Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms, including bacteria, yeast and fungi, that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., 2001). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., 2001). The effective utilization of cellulose through biological processes is one approach to overcoming the shortage of foods, feeds, and fuels (Ohmiya et al., 1997).
Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into three major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”), and beta-glucosidases (β-D-glucoside glucohydrolase; EC 3.2.1.21) (“BG”). (Knowles et al., 1987; Shulein, 1988). Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose (Nevalainen and Penttila, 1995). Beta-glucosidase acts to liberate D-glucose units from cellobiose, cellooligosaccharides, and other glucosides (Freer, 1993).
Cellulases are known to be produced by a large number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system capable of degrading crystalline forms of cellulose, such that the cellulases are readily produced in large quantities via fermentation. Filamentous fungi play a special role since many yeast, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose. (See, e.g., Aro et al., 2001; Aubert et al., 1988; Wood et al., 1988, and Coughlan, et al.)
The fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification. For example, multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources including Trichoderma reesei which contains known genes for 2 CBHs, e.g., CBH I (also known as Cel7A or glycosyl hydrolase family (GH)7A) and CBH II (also known as Cel6A or GH6A), a number of EGs, e.g., EG I (also known as Cel7B or GH7B), EG II (also known as Cel5A or GH5A), EG III (also known as Cel12A or GH12A), EGV (also known as Cel45A or GH45A), EGVI (also known as Cel74A or GH74A), EGVII (also known as Cel61B or GH61 b) and EGVIII, and a series of BGs, e.g., BG1, BG3, and BG5.
In order to efficiently convert crystalline cellulose to glucose, a complete cellulase system comprising components or enzymatic activities from each of the CBH, EG and BG classifications is typically required, with isolated components less effective in hydrolyzing crystalline cellulose (Filho et al., 1996). A synergistic relationship has been observed amongst cellulase components from different classifications. In particular, the EG-type cellulases and CBH-type cellulases synergistically interact to more efficiently degrade cellulose. (See, e.g., Wood, 1985.).
Cellulases are known in the art to be useful in the treatment of textiles for the purposes of enhancing the cleaning ability of detergent compositions, for use as a softening agent, for improving the feel and appearance of cotton fabrics, and the like (Kumar et al., 1997).
Cellulase-containing detergent compositions with improved cleaning performance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and 2,094,826) and for use in the treatment of fabric to improve the feel and appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757; GB App. No. 1,358,599; The Shizuoka Prefectural Hammamatsu Textile Industrial Research Institute Report, Vol. 24, pp. 54-61, 1986), have been described.
Cellulases are further known in the art to be useful in the conversion of cellulosic feedstocks into ethanol. This process has a number of advantages, including the ready availability of large amounts of feedstock that is otherwise discarded (e.g., burning or land filling the feedstock). Other materials that consist primarily of cellulose, hemicellulose, and lignin, e.g., wood, herbaceous crops, and agricultural or municipal waste, have been considered for use as feedstock in ethanol production. In recent years, new classes of glycosyl hydrolases have been identified that provide further auxiliary effects that enhance or augment the enzymatic hydrolysis of cellulosic materials, although the mechanisms of action of many of these new auxiliary enzymes have not been fully elucidated. One such family of glycosyl hydrolases, which had earlier been annotated as GH61 family (see, e.g., Harris et al. “Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family” Biochemistry 2010, vol. 49, pp. 3305-3316), had been repeatedly re-annotated, most recently to Auxiliary Activity (AA) Family 9 after the discovery that some family members are lytic polysaccharide monooxygenases (Levasseur A. et al, “Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes” Biotechnol Biofuels 2013, vol 6, issue 1, pp. 41). At least two GH61 enzymes are present in the T. reesei (Saloheimo M., “cDNA cloning of a Trichoderma reesei cellulase and demonstration of endoglucanase activity by expression in yeast” Eur J Biochem. 1997 vol. 249, issue 2: pp. 584-91; Karlsson et al., Homologous expression and characterization of Cel61A (EG IV) of Trichoderma reese?′ Eur. J. Biochem. 2001 vol. 268, pp. 6498-6507; Karkehabadi et al., “The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution” J Mol Biol. 2008, vol. 383 issue 1: pp 144-154; Martinez et al., “Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)” Nature Biotechnology 2008, vol. 26, pp. 553-560). In the very recent past, it was reported that up to four more of these glycosyl hydrolases have been identified in the Trichoderma reesei genome (Hakkinen M. et al, “Re-annotation of the CAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates” 2012, Microb Cell Fact. Vol 4, issue 11, pp. 134).
It would be an advantage in the art to provide a set of GH61 enzyme variants with improved capacity, when combined with one or more cellulases, and optionally also one or more hemicellulases, to augment the efficacy and efficiency of hydrolyzing lignocellulosic biomass substrates to monosaccharides, disaccharides, and polysaccharides. Improved properties of the variant GH61 polypeptide include, but are not limited to: altered temperature-dependent activity profiles, thermostability, pH activity, pH stability, substrate specificity, product specificity, and chemical stability.